Platelet ?IIb?3 plays a central role in hemostasis, but also contributes to thrombosis. ?V?3 has wider tissue distribution and is implicated in contributing to the pathogenesis of osteoporosis, sickle cell disease, tumor angiogenesis, viral invasion, fibrosis, tumor angiogenesis, and supravalvular aortic stenosis. We have developed novel ?IIb?3 pure antagonists that lock the receptor in the inactive conformation in contrast to existing RGD- based antagonists that induce the receptor to undergo a major conformational change that places it in a high affinity ligand binding state and thus are partial agonists. We have brought one of our pure antagonists (RUC-4) forward for clinical development. We then built on this experience to synthesize non-peptide, small molecule, orally available, potent pure antagonists of ?V?3. We also provided the highest resolution 3D reconstruction of intact ?IIb?3 in a nanodisc that unexpectedly demonstrated an upward tilt of the headpiece and separation of the leg regions. In a search for ancillary binding site(s) on ?IIb?3 and fibrinogen beyond the interaction between the C-terminal ?-chain peptide ?404-411 with the RGD-binding site on ?IIb?3, we demonstrated that activated, but not unactivated, ?IIb?3 could mediate adhesion to fibrinogen fragment D98, which lacks ?404-411. In the upcoming grant period we will bring advanced computational and cryo-electron microscopy (cryo-EM) analysis in collaboration with Drs. Marta Filizola and Thomas Walz to extend these studies to address important unresolved aspects of ?IIb?3 ligand binding and initiate translational studies with our new ?V?3 antagonists. Specific aim 1. To provide further insight into ?IIb?3 receptor activation and ligand binding: a. Identify ancillary interactions between the fibrinogen ? module and ?IIb?3 beyond the interaction of ?- 404-411 with the RGD binding pocket by: 1) Functional ligand binding assays, 2) Molecular modeling, 3) Cryo- EM, 4) High throughput screening to identify small molecule and monoclonal antibody (mAb) inhibitors. b. Use cryo-EM and computational analyses to: 1) Improve the resolution of our 3D reconstruction of unliganded, full length purified ?IIb?3 in a nanodisc lipid bilayer, 2) Obtain additional 3D models of ?IIb?3 activated by the talin head domain (THD) and THD-activated, fibrinogen-liganded ?IIb?3, 3) Define the activation pathway and transformation among the inactive receptor; the inactive, unliganded receptor; and the active, liganded receptor. Specific aim 2. To develop and test the biologic effects and potential clinical use of novel pure ?V?3 antagonists: a. Further characterize the pure ?V?3 antagonists we have synthesized for specificity for different ?V- containing receptors, binding characteristics, and cellular effects, and make them available as tool compounds. b. As a prelude to potential clinical testing, assess the species specificity of the pure ?V?3 antagonists, and then compare their impact to those of current RGD-based partial agonists on the pathophysiology of animal models of disease.